Pyrus ussuriensis Maxim 70% ethanol eluted fraction ameliorates inflammation and oxidative stress in LPS‐induced inflammation in vitro and in vivo

Abstract Pyrus ussuriensis Maxim (PUM) is a popular fruit among consumers, and also used as medical diet for dissolving phlegm and arresting cough. The present study aims to investigate the potential protective effect of P. ussuriensis Maxim 70% ethanol eluted fraction (PUM70) on lipopolysaccharide (LPS)‐induced alveolar macrophages and acute lung injury (ALI) in mice. A total of 18 polyphenol compounds were tentatively identified in PUM70 by mass spectrometry (MS) analysis. The results in vivo suggested that PUM70 treatment could effectively alleviate the histological changes, and significantly inhibit the activity of myeloperoxidase (MPO) and the expression of pro‐inflammatory cytokines (tumor necrosis factor‐α (TNF‐α), interleukin‐1β (IL‐1β), and interleukin‐6 (IL‐6)). The cell test results show that PUM70 exerted its protective effect by suppressing the messenger RNA (mRNA) expression levels (inducible nitric oxide synthase (iNOS) and cyclooxygenase‐2 (COX‐2) and decreasing nitric oxide (NO) and prostaglandin 2 (PGE2) contents. In addition, it also inhibited the overproduction of pro‐inflammatory cytokines (TNF‐α, IL‐1β, and IL‐6). Furthermore, PUM70 induced the production of heme oxygenase 1 (HO‐1) protein and nuclear translocation of Nrf2 (nuclear factor erythroid 2‐related factor 2), indicating that PUM70 could mitigate oxidative injury via the Nrf2/HO‐1 pathway. Moreover, PUM70 inhibited LPS‐induced inflammation by blocking the phosphorylation of mitogen‐activated protein kinases (MAPKs). The above results indicate that PUM70 has protective effects on LPS‐induced ALI, possibly be related to the inhibition of MAPK and Nrf2/HO‐1 signaling pathways.

The release of a large number of inflammatory cytokines, such as interleukin-1β (IL-1β), tumor necrosis factorα (TNFα), and so on, has been proved to be one of the key factors promoting the development of ALI (Lei et al., 2018). In recent years, the development of ALI drugs has been mainly targeted at the control of degree of inflammation . Numerous studies have been reported that oxidative stress is one of the main causes of inflammatory response, which could cause lesions to DNA, proteins, and lipids by regulating antioxidant enzyme production (Kwon et al., 2018;Yang et al., 2016).
Therefore, some antioxidant pathways potentially mediated by endogenous or exogenous compounds may be utilized as the therapeutic targets to address ALI (Kim et al., 2020).
Naturally active ingredients such as flavonoids and polyphenols have strong anti-inflammatory effects with high safety and effectiveness, and can simultaneously act on multiple targets in the inflammatory pathway (Li et al., 2016;Qian et al., 2019). Pyrus ussuriensis Maxim (Anli in Chinese) is an important nutritional and economic fruit crop in China, which has been used as medical diet for dissolving phlegm and arresting cough. In our previous study, the extract of P. ussuriensis Maxim was found to contain high concentrations of both total phenolics and pentacyclic triterpenoids (Peng et al., 2020). However, in spite of the promising results observed in the anti-inflammatory and antioxidant activity of this pear, researches in this field are still scarce. Therefore, to be included in the market for these uses, more studies focused on their specific effects in vitro and in vivo are needed. In this work, we investigated the potential protective effect of P. ussuriensis Maxim extract against LPS-induced alveolar macrophages and ALI in mice.

| Collection of plant materials and preparation of extracts
The P. ussuriensis Maxim (PUM) fruits were collected on their commercial maturity in September 2019, from Qinhuangdao City, Hebei Province, China, and kept at 4°C before use. The extraction was conducted according to the methods of our previous study (Peng et al., 2018). In brief, the ethyl acetate fraction was further purified using microporous resin with an ethanol-water gradient (0, 100 to 90: 10, v/v). The 70% ethanol eluted fraction was collected and freeze-dried (Alpha 2-4 Dplus, Christ, Germany) to obtain PUM70.

| Analysis of PUM70
The identification of components in PUM70 was performed using Ultra Performance LC system (Thermo Fisher U3000, USA) and Exactive™ mass spectrometer equipped (Thermo Fisher Scientific, USA). The data were subsequently input into the Mass Frontier 7.0 software and combined with ChemSpider network database. The total ion current chromatograms for PUM70 are shown in Figure S1.

| Animals
Prior to the experiment, experimental use of animals was approved by the Ethics Committee of Hebei Normal University of Science and Technology. Male BALB/c mice (20-24 g) were purchased from Vitong Lihua Laboratory Animal Technology Co., Ltd (Beijing, China). The mice were kept in a clean animal house and fed with sterile food and water. During the test period, the temperature and relative humidity of the environment were kept at 20-24°C and 40%-70%, respectively, with a light-and-darkness cycle of 12 h daily. After a period of acclimatization, the mice were randomly divided into control group, LPS group, dexamethasone (5 mg/kg) group, and PUM70 (50, 100, and 200 mg/kg) groups, eight for each group. Equal volumes of normal saline (control group and LPS group), dexamethasone, and PUM70 were administered intragastrically once a day. On the seventh day, the mice were anesthetized and fixed, then LPS (10 μg LPS was dissolved in 50 μl phosphate-buffered saline (PBS)) was injected intratracheally through a microsprayer needle (model IA-1C, Penn-Century, USA) attached to a microsyringe (FMJ-250, Penn-Century, USA) to establish the experimental groups, or injected with sterile water to serve as the control group. After the induction of injury for 6 h, the mice were sacrificed. The blood and lung tissues were collected and stored at −80°C.

| Histopathological observation
The upper lobe tissues of the right lung were soaked in 4% formaldehyde solution and fixed for 48 h, then dehydrated with ethanol, made transparent with xylene, dipped in wax, embedded, sliced (5 μm), retrieved, and dried. Sections were stained with hematoxylin-eosin (HE) and observed under an inverted microscope.

| Inflammatory index in lung
The bronchial tubes were flushed with 2 ml cold PBS buffer three times to collect the bronchoalveolar lavage fluid (BALF). One part of BALF was taken to test the protein content with the BCA protein assay kit, and the other part of BALF was taken to measure the proinflammatory cytokine content according to the ELISA kit instruction. The neutrophil count was determined on a smear prepared by Wright-Giemsa stain.

| Oxidative stress index in the lung
The left lung tissue was taken and rinsed with PBS and then ground with an abrasive rod. The cell suspension was filtered with 300 μm Nilon net and added with fluorescent probe dichlorodihydrofluorescein diacetate (DCFH-DA). The cell precipitate was collected after centrifugation and washed with PBS twice to be used for flow cytometry. The results were shown as the average fluorescence intensity value (the fluorescence intensity in each cell). The lower lobe of the right lung was ground with liquid nitrogen to prepare the tissue homogenate. MDA, SOD, GSH, and MPO were detected, respectively, according to the reagent instructions.

| Cell culture and cell viability
The mouse alveolar macrophages (MH-S) were obtained from the Procell Life Science & Technology Co., Ltd. (Wuhan, China).
Subsequently, cell culture and cell viability tests were determined following the reported method (Kwon et al., 2018). Briefly, the MH-S cells (1 × 10 5 cells per well) were seeded into a 96-well plate. After 24 h of incubation at 37°C, 10 μl of PUM70 at various concentrations was added to each well of the plates. After incubation for additional 24 h, the PUM70-treated cells were incubated with 100 μl MTT (0.5 mg/ml) for 4 h. After the removal of supernatant, MTT-formazan was dissolved in 200 μl dimethyl sulfoxide (DMSO). A microplate reader (ELX-800, BioTek, Vermont, USA) was used to measure the absorbance at 544 nm ( Figure S2).

| Oxidative stress index in cells
The cells were lysed with RIPA (radioimmunoprecipitation assay) lysis buffer and then the supernatant was collected after centrifugation for the detection of oxidative stress index. MH-S cells were treated with PUM70 (2.5, 5, and 10 μg/ml) for 2 h and stimulated with 2 μg/ml LPS for 1 h. Then, the cells were incubated with 10 M DCFH-DA for 30 min at 37°C. After centrifugation, the precipitates were collected and washed twice with PBS. The intracellular ROS content was analyzed by flow cytometry (CytoFLEX LX; Beckman Coulter, USA) and spectrofluorometer.

| Inflammatory index in cells
MH-S cells (1 × 10 5 cells/well) were pretreated with PUM70 (in gradient concentrations) for 2 h, treated with 2 μg/ml of LPS, and incubated for 24 h. After centrifugation, the supernatant was collected.
The cell lysis solution was mixed with Griess reagent and incubated for 15 min. The absorbance was measured at 540 nm by the Epoch microplate reader (PerkinElmer, USA), and the level of NO was calculated according to the standard curve. For the PEG2 assay, cells were pretreated with PUM70 (in gradient concentrations) for 2 h, treated with 2 μg/ml of LPS, and incubated for 24 h. After centrifugation, the supernatant was collected. The levels of PGE2 and pro-inflammatory cytokines were measured with the ELISA kit.

| qRT-PCR
The quantitative reverse transcription-polymerase chain reaction (qRT-PCR) assay was performed according to the reported method . Relative fold-changes were calculated according to 2 −ΔΔCT .

| Western blotting
MH-S cells were prepared in petri dishes 12 h prior to the assay and treated with PUM70 for 24 h, followed by stimulation with LPS (2 μg/ ml) for 1 h. The western blotting was performed in accordance with Kwon et al. (2018). Cytoplasmic proteins and nuclear proteins were fractionated using a CelLytic™ NuCLEAR™ Extraction Kit. Pierce BCA Protein Assay Kit was used to determine the protein concentration. The extracted samples were fractionated by 10% sodium dodecyl sulfate/polyacrylamide gel electrophoresis (SDS/PAGE) and transferred to Immunoblot polyvinylidene difluoride (PVDF) films.

| Statistical analysis
The SPSS 20.0 version (SPSS Inc., Chicago, IL, USA) was used to analyze the Pearson correlation coefficients. The significant differences data were performed for comparative analysis using Duncan's multiple range test. Statistically, p < .05 indicated a significant difference.

| Pathological effect of PUM70 in ALI mice
As shown in Figure 1, there was no significant pathological change in the lung tissue of the normal control group. However, significant pathological changes of the lung tissues appeared in the LPS group, such as alveolar volume decrease, alveolar septum thickening, and extensive inflammatory cell infiltration. Interestingly, the pathological changes of mice were greatly alleviated in each PUM70 group, including the decreases in inflammatory cells, bronchoalveolar wall thickness, and pulmonary congestion (Figure 1). With increasing PUM70 concentration, the inhibitory effect tended to become more significant.

| Effects of PUM70 on oxidative stress index in the lung
The flow cytometry results showed that the average fluorescence value of ROS was significantly increased in the LPS treatment group (p < .001), while pretreatment with PUM70 could effectively reduce ROS (p < .01) (Figure 3a). Similarly, the LPS group had significantly decreased SOD activity and GSH content, while increasing MDA content than the control group. However, the situation had significantly improved after PUM70 treatment (Figure 3b-d).

| Effect of PUM70 on the oxidation index of LPS-induced alveolar macrophages
The elevation of ROS level caused by changes in different signaling pathways facilitates the formation of a pro-inflammatory microenvironment and promotes the progress of disease development (Kwon et al., 2018). As can be seen from Figure

| Effect of PUM70 on the expression of HO-1 and Keap1/Nrf2
Nrf2/HO-1 is an important endogenous antioxidant pathway. Here, western blot was performed to investigate the impact of PUM70 on the expression of HO-1, Nrf2, and Keap1 (Kelch-like ECHassociated protein 1) induced by LPS (Figure 6a). The data demonstrated that PUM70 treatment significantly (p < .01) increased the intranuclear levels of HO-1 and Nrf2 in the cells relative to the LPS group (Figure 6b,d). In addition, PUM70 treatment dramatically rescued the upregulation of cytosolic Keap1 caused by LPS stimulation (Figure 6c).

| Effects of PUM70 on the phosphorylation levels of MAPKs
The results in Figure 7 indicate that there were significant increases in the phosphorylation of three MAPKs (p38, ERK (extracellular signal-regulated kinase), and JNK (c-Jun N-terminal kinase)) in the LPS treatment group. However, the phosphorylation of MAPKs in the PUM70 treatment group was significantly inhibited, and the alleviating effect was correlated with the dose of PUM70 (Figure 7b-d).

| DISCUSS ION
Acute lung injury (ALI) is a common disease in clinical practice, whose specific molecular mechanism remains unclear due to its complexity and diversity. Changes in inflammatory response and vascular permeability generally occur at the initial stage of ALI (Zambelli et al., 2012). At the early stage of ALI, there will be an increase in the microcirculation permeability of lung tissue, leading to the extravasation of a large amount of plasma and proteins from blood vessels into the lung interstitium and alveolar cavity, and finally causing noncardiogenic pulmonary edema (Matthay et al., 2012). This is a pathologic feature of ALI and an important cause of progressive dyspnea and hypoxemia in patients. Therefore, a timely control of disease progression at the early stage of ALI is essential to the increase of cure rate. Recent studies have shown that some natural phytochemicals could inhibit inflammation by blocking the release of inflammatory cytokines and reduce the infiltration of pro-inflammatory macrophages (Akanda et al., 2018;Raso et al., 2001). Numerous studies have revealed the biological effects of pear fruit, such as antiinflammatory and antioxidant effects, which can be mainly ascribed to the abundant phenolics in the fruit (Li et al., 2012). In this work, 18 phenolic compounds were identified in PUM70 using UPLC-MS/MS (ultraperformance liquid chromatography tandem mass spectroscopy), including flavan-3-ols, phenolic acids, anthocyanins, and flavonols. It has been reported that some phenolic compounds, such as kaempferol, quercetrin, and isoquercitrin, could prevent a variety of inflammatory diseases by activating the Nrf-2 pathway (Comalada, et  to have strong physiological activity, particularly in terms of antiinflammatory activity (Hong et al., 2015). As caffeic acid derivatives, caffeoylquinic acid activates Nrf2 and inhibits NF-κB activation to prevent oxidative stress . Therefore, the excellent antioxidant and anti-inflammatory activity of PUM70 in this study may be closely related to these phenolic compounds.
It has been reported that ALI was associated with neutrophilic infiltration and increased inflammatory cytokines and exudation of proteins (Butt et al., 2016). At the early stage of ALI, neutrophils congregated at the site of lung injury to protect the host. However, excessive activation of neutrophils will cause damage to the tissue due to the release of various bioactive substances such as inflammatory cytokines and proteases (Grommes & Soehnlein, 2011).
Numerous studies have shown that the decrease in neutrophils is related to the prognosis of ALI (Aulakh, 2018). Moreover, the release of pro-inflammatory cytokines will activate neutrophils and further cause neutrophil infiltration, which may damage vascular epithelium and endothelial cells, followed by the occurrence of pulmonary edema due to the infiltration of plasma proteins into the interstitium of the lungs (Sharp et al., 2015). The excessive accumulation of neutrophils can also cause oxidative stress. ROS, as major oxidative components, is closely related to multiple signaling pathways triggered by inflammation-related signal transduction cascades (Li & Engelhardt, 2006). It has also been revealed that ROS are potentially involved in the activation of nuclear factor-kappa B (NF-κB) signaling pathway at the early phase of several diseases (Nakajima & Kitamura, 2013). Thus, it is important to inhibit the overproduction of ROS for alleviating the oxidative stress caused by inflammatory cells. In addition, MDA could damage cell membranes, while antioxidant enzymes such as SOD and GSH can mitigate oxidative stress damage (Annapurna et al., 2013). This study confirmed that PUM70 can alleviate the pathological changes and oxidative stress injury of lung tissue. As another possible mechanism, LPS-induced oxidative stress in ALI can be inhibited by the activation of Keap1/Nrf2/HO-1 signaling pathway (Li et al., 2014). Nrf2 is normally sequestered by Keap1 in the cytosol. However, certain stimuli can trigger the release of Nrf2 from Keap1, facilitating the translocation of Nrf2 into the nucleus to activate HO-1 (Balogun et al., 2003). HO-1, an antioxidant enzyme, plays a critical role in preventing ROS damage to macrophages, and therefore has been intensively studied as a promising target in antioxidant studies (Araujo et al., 2012). Previous studies have shown that Nrf2 knockout mice are highly susceptible to oxygeninduced lung injury and lung epithelial cell death (Cho et al., 2002;Papaiahgari et al., 2004). Under the nonactivated state, Nrf2 is using Nrf2-knockout mice. The present study provides a foundation for the application of PUM70 as a potential anti-inflammatory ingredient in functional foods or nutraceutical formulations.

ACK N OWLED G M ENT
The authors gratefully acknowledge Engineering Research Center of Chestnut Industry Technology of Ministry of Education for providing testing equipment.

FU N D I N G I N FO R M ATI O N
This research was funded by Hebei Province Science and Technology Support Program, grant number 20322803D.

CO N FLI C T O F I NTE R E S T
The authors declared no conflicts of interest.

DATA AVA I L A B I L I T Y S TAT E M E N T
The data that support the finding of this study are available from the corresponding author upon reasonable request.

E TH I C S S TATEM ENT
The animal study protocol was approved by the Ethical Committee for the Experimental use of animals at Hebei Normal University of Science and Technology (no. L2020109).